Análise funcional do gene que codifica xiluloquinase em Xanthomonas citri

Abstract

Citrus canker causes a decrease in the productivity and quality of citrus fruits due to the lack of efficient control and eradication measures and is caused by the bacterium Xanthomonas citri subsp citri (XAC). In proteomics studies previously carried out by our research group, the involvement of xylose metabolism, a carbohydrate present in the plant wall, in the bacterial infection process was bserved, demonstrating that studies with XAC may have biotechnological importance for the use of plant biomass. The objective of this work was to characterize the XAC xylulokinase (XK) gene, one of the enzymes belonging to this metabolic pathway, responsible for the phosphorylation of D-xylulose [produced from xylose by xylose isomerase enzyme (XI)] to D-xylulose-5-phosphate, then allowing xylose to be metabolized via the pentose phosphate pathway. There are two ORFS present in the XAC genome annotated as coding for XK (XAC1775 or xylB1 and XAC4244 or xylB2), and the alignment of the two amino acid sequences revealed only 26% similarity between them. These two genes annotated as coding for XK were used in the construction of an IPTG-inducible heterologous expression system to evaluate their predicted activities. The expression of the xylB2 gene returned an insoluble protein, and studies with xylB1 continued. Enzyme activity studies demonstrated that xylB1 encodes a functional xylulose kinase enzyme, and kinetic studies revealed the value of the Michaelis-Menten constant (Km) to be 0.7947 ± 0,4032 mM and the value of Vmax to be 0,01217 ± 0,001130 µMol of xylulose.min-1.mg of protein-1. In a Pull-Down assay to investigate possible XK interacting proteins, the results indicated absence or undetectable concentrations of proteins from the total cell extract that interact with XK of XAC. An attempt was also made to obtain a mutant strain deleted in the xylB1 gene, based on a double homologous recombination methodology, with the construction of the suicide vector (pNPTS138_ xylB1) with the in tandem cloning of fragments 1 kb upstream and 1 kb downstream to the xylB1 gene. So far, a heterogeneous colony was obtained, possibly containing the wild and the mutant strains (mutant to be named XAC∆1775), which is in the process of isolation of the mutant for further characterization against the wild strain regarding its pathogenicity. Given the importance of xylose metabolism within the current economic-strategic context of using plant biomass, this work aims to add knowledge about this metabolic pathway in XAC and is a pioneer in the molecular characterization of the xylulokinase (XK) gene in the genus Xanthomonas.